Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Abstract

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert Ò and molecular. Enzymatic evaluation was performed using the mycoalert Ò kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert Ò method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (\20 min).

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

“…In this study, the false negative results for enzymatic method were observed in only two cell lines i.e., PEER (NCBI C511) and SH-SY5Y (NCBI C611). In the other 28 cases, the mycoalert Ò results were concordant with the real-time PCR results (Mariotti et al 2008;Markoullis et al 2009;Volokhov et al 2011;Molla Kazemiha et al 2014).…”

“…These cell lines were incubated at 37°C in 88 % humidified and 5 % CO 2 atmosphere. The culture medium for each cell line was prepared according to the recommended instructions and supplemented by 10-20 % fetal bovine serum (Sigma, Deisenhofen, Germany) and growth factors as described previously (Molla Kazemiha et al 2011Kazemiha et al , 2014. In order to detect the mycoplasma contamination after harvesting, each cell line was cultured in an antibiotics-free medium for a week (at least 4-5 days) without exchanging the medium (Phelan 2006(Phelan , 2007 (Barile and McGarrity 1983;Rottem et al 2012).…”

“…A rapid screening method has been provided by these enzymes for sensitive detection of mycoplasma contamination in cell cultures (Pitt et al 2012). In the present study, mycoplasma detection was conducted by measuring the acetate kinase or carbamate kinase activity as described previously (Mariotti et al 2008;Cheong et al 2011;Molla Kazemiha et al 2014). Mycoplasma detection using conventional PCR method Extraction of DNA from suspicious samples was performed for PCR with specific universal primers.…”

“…The cell pellet was rinsed with 70 % ethanol and dried for 30 min at 25°C. Finally, the suspension of precipitated DNA was made in RNA-DNA free sterile deionized water and maintained at -20°C (Molla Kazemiha et al 2011Kazemiha et al , 2014). …”

“…In addition to 11 species-specific primer pairs, a universal primer pair was designed based on 16S rRNA mollicutes to detect the mycoplasma contamination of cell lines with PCR method which is indicated in Table 2 (Molla Kazemiha et al 2009Kazemiha et al , 2011Kazemiha et al , 2014. Master mix of PCR was prepared by 2.5 ll PCR 10 9 Buffer, 1 ll Taq DNA polymerase enzyme (1 U), 1 ll dNTPs (50 lM), 1 ll magnesium chloride (1.5 mmol), 3 ll forward and reverse primers (15 pmol), 12.5 ll distilled water and 1 ll mycoplasma genomic DNA (0.1 lg/ll) or cell DNA (1 lg/ll).…”